Microchip device for cell lysis, multiplex PCR amplification, and electrophoretic sizing.
The steps of cell lysis, multiplex PCR amplification, and electrophoretic analysis are executed sequentially on a monolithic microchip device. The entire microchip is thermally cycled to lyse cells and to amplify DNA, and the products are then analyzed using a sieving medium for size separation and an intercalating dye for fluorescence detection. Using a standard PCR protocol, a 500-base pair (bp) region of bacteriophage lambda DNA and 154-, 264-, 346-, 410-, and 550-bp regions of E. coli genomic and plasmid DNAs are amplified. The electrophoretic analysis of the products is executed in <3 min following amplification using hydroxyethyl cellulose or poly(dimethylacrylamide) sieving gels. Product sizing is demonstrated by proportioning the amplified product with a DNA sizing ladder.
Waters
L C
LC
Oak Ridge National Laboratory, Tennessee.
Jacobson
S C
SC
Kroutchinina
N
N
Khandurina
J
J
Foote
R S
RS
Ramsey
J M
JM
D004269:DNA, Bacterial / Q000096:biosynthesis / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008838:Microcomputers; D016133:Polymerase Chain Reaction / Q000295:instrumentation*
D004269:DNA, Bacterial